Pharmaceutical Formulations and Ligands for Use Therein; Mimetics for UEA-1

ABSTRACT

UEA-1 Mimetics, pharmaceutical formulations comprising them, and their uses as targeting agents for therapeutic and diagnostic purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/302,822, filed Jul. 2, 2001 and U.S. provisional application Ser.No. 60/302,868, filed Jul. 3, 2001, both of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention has to do with pharmaceutical formulations, andparticularly pharmaceutical formulations suitable for enteraladministration, notably oral dosage forms. The invention has particularreference to adapting a pharmaceutical formulation with a view toimproving the take-up of pharmaceutically-active ingredient such as adrug or vaccine through the body's epithelial layer, especially theenterocytes lining the lumenal side of the gastrointestinal tract (GIT).An aspect of the invention relates to the identification, preparationand use of categories of compounds, including novel compounds, able tobe incorporated in a pharmaceutical formulation to enhance the transportof pharmaceutically-active ingredients through the epithelial layer.Such compounds include novel compounds useful for other purposes asmimetics of certain naturally-occuring compounds.

This invention also has to do with compositions and methods useful inthe diagnosis and prognosis of disease states, in particular involvingthe imaging e.g. by staining, or marking, of certain tissue cell typesfrom the human or animal body in order to help establish the status orcondition of the tissue. In particular in one aspect the compositionsdisclosed herein are applied to the investigation or evaluation of cellsof the gastrointestinal tract (GIT). This may be for assessment of asuspected or known condition of inflammation, neoplasia, dysplasia orother abnormal and perhaps malignant cell transformation in the cellsconcerned. Diseases of particular interest include colon carcinoma,ulcerative colitis and Crohn's Disease.

In another aspect the composition and methods disclosed herein areapplicable to the evaluation of the disposition of blood vessels inhuman or animal tissue, wherever malignant or non-malignant.

BACKGROUND TO THE DRUG/VACCINE DELIVERY ASPECTS OF THE INVENTION

It is well known that the effect of a pharmaceutically-active ingredientadministered to the body depends greatly on the administration route.Ideally of course one wants to create a concentration of the activeingredient localised at the affected site, but there is seldom apractical way of achieving this directly. For many drugs parenteraladministration (e.g. intravenous, subcutaneous, intramuscular) is mosteffective but it has well known limitations and disadvantages. Theseinclude the risk of adverse effects from local high concentrations ofdrug substance in the body, the risk of infection at injection sites andin general a measure of discomfort or inconvenience tending to reducepatient compliance. Patient compliance is very important where a drug isto be routinely self-administered.

Other routes exploit drug transport across epithelial barriers, e.g.buccal, nasal, vaginal, rectal and intestinal. Among these, enteral andparticularly oral administration is by far the most convenient andfavoured by patients. However enteral drug delivery is notoriouslyproblematic because of the very indirect route by which the activeingredient enters the system. To show a therapeutic effect anorally-administered drug must survive the acidic environment of thestomach and then cross the epithelial barrier i.e. the gut lining inorder to enter the circulation or interact with the immune system.

A number of published and practical proposals exist for coating and/orencapsulating pharmaceutically-active ingredients in excipients whichallow the active substance to pass through the stomach and survive untilthey reach the target region of the GIT. One formulation type ofparticular current interest is the so called microparticles andnanoparticles, made of bioerodible or biodegradable polymeric excipientswhich can retain and protect the active substance as it travels alongthe GIT and then be absorbable through the gut wall, after which theparticles should break down in the bloodstream and release the activeingredient to exert its therapeutic effect.

In practice however it has been found that bioavailability with theseformulations is nevertheless much lower than with parenteral routes andalso highly variable from one patient to another. This is generallyregarded as being because of the difficulty in getting the activesubstance, in its bioerodible/biodegradable encapsulation where present,across the gut wall with its mucosal layer and highly selectiveepithelial cells.

Particular challenges in this respect arise in relation to thepharmaceutical use of biological or biotechnology products such ashormones and enzymes. These are generally macromolecular, e.g. proteins,peptides, genes, pieces of DNA, DNA vaccines, antisense oligonucleotidesetc. Their large molecular size makes it difficult for them to cross theepithelium. Their stability in the GIT is poor because of the action ofacids and enzymes. The bioavailability via the oral route is therefore avery low percentage, which is doubly problematic having in mind suchdrugs' scarcity and expense. Currently only parenteral administration isusable, with its attendant disadvantages. It would be highly desirableto improve the bioavailability of these macromolecular drugs andvaccines via other routes.

Various proposals have been published relating to means for givingdrug-active particles a positive affinity to the gut wall so thatwhatever transepithelial mechanism operated would have a persistentpresence of the active substance to work on, and/or some biochemicalincentive to promote cellular uptake of the active.

Some work has been done on this and it has been pointed out and shownthat various lectins—naturally-occurring protein substances withspecific affinities for certain sugar residues—will bind specifically tomodel enterocyte-type cell lines. This is because the enterocyte surfacedisplays oligosaccharide moieties. It has therefore been proposed to uselectins as carriers for oral drug delivery, particularly taking intoaccount that non-toxic plant lectins are already in the human diet.Reference is made to the following publications. F. Gabor et al, Journalof Controllable Release 55 (1998), pp 131-142: N. Foster et al, Vaccine16, No. 5 (1998), pp 536-541: C. M. Lehr et al, Pharm Res. 12 (1992) pp547-553, and other articles on related themes.

Despite these interesting results, the use of lectins to promote“bioadhesion” of drug substances in the GIT remains problematic, becausesuch large protein molecules are liable to degradation and loss ofactivity both in the gut under the action of enzymes and duringprocessing to prepare formulations. This large size, together withpotential immunogenicity and cytotoxicity effects, limits the use oflectins per se as targeting agents to deliver drugs and vaccines to andacross the human GIT.

The present inventors have carried out very extensive investigationswith a view to identifying, testing and preparing alternative substancesshowing an affinity for epithelial cells, and hence a “bioadhesive”capacity making them useful as moieties, ingredients or coatings inenterally-administered pharmaceutical formulations.

BACKGROUND TO THE DIAGNOSTIC AND PROGNOSTIC ASPECTS OF THE INVENTION

It has been noted that alteration and/or upregulation of surface sugarresidues in the intestinal mucosa have been associated with malignanttransformation, dysplastic changes and extensive colitis. For example,tissues from ulcerative colitis and Crohn's disease patients exhibitedaltered distribution of Ulex europaeus I (UEA1) labelling sites(Yoshioka et al, 1989). The expression of lectin-binding sites on humanintestinal goblet mucin was specifically altered in these conditions,thus possibly providing an alternative approach to the assessment ofneoplastic risk in these diseases (Yoshioka et al, 1989). Patterns ofUEA1 and Dolichos biflorus agglutinin (DBA) in carcinomas of the largeintestine were also altered when compared to normal mucosa and adenomas(Iwakawa et al, 1996)

UEA1 is a lectin protein of approximately 60 kDa derived from furze(Ulex europaeus) that is known to bind to fucose residues and inparticular is known to bind to epithelial cells.

We have done a large amount of work investigating the properties of UEA1and in identifying and synthesising other molecules which mimic UEA1, inthe sense that they share to a lesser or greater degree (a greaterdegree, in some cases) the characteristic binding activity of UEA1 toepithelial cells, but because of their simpler molecular structure mayenjoy any of higher stability, reduced cost, easier labelling or thepossibility of use in multivalent forms. These other molecules, referredto in what follows as “UEA1 mimetics” may have any of a variety oforganomolecular structures. They may be peptides, peptidomimetics and/orsmall organic molecules. A variety (non-limiting) of such molecules andmethods of identifying and preparing them are discussed later below.

We have confirmed the effectiveness of UEA1 and of its mimics in bindingto human intestinal tissue sections. In view of the relationships notedabove between various disease states, we put forward the first aspect ofthe present invention which is methods and compositions for assessingthe status of GIT cells by means of imaging, using UEA1 or a UEA1mimetic as a localisation agent which binds characteristically toepithelial cells.

A second aspect of the invention relates to the fact that UEA1 bindsspecifically to the vascular endothelium of various human tissuesirrespective of the blood group type or secretive status of the tissue.UEA1 staining of blood vessels has been evaluated in various studies ofmalignant and nonmalignant tissues. For example, most vessels inmalignant and nonmalignant tissues of bladder, prostate and testis werereadily identified (Fujime et al, 1984). UEA1 visualized the endotheliaof blood vessels with equal intensity, sensitivity, and reliability innormal brain and in tumour tissue with neovascularization (Weber et al,1985). While large, medium, and small vessels were equally welldemonstrated by UEA1 and antibodies against FVIII/RAG, capillaries andendothelial sprouts were stained more consistently and intensely byUEA1. UEA1 was also a specific and sensitive marker for the endothelialcells in benign vascular lesions (Miettinen et al, 1983). UEA1 alsostained many neoplastic cells of endothelial sarcomas. Melanomas,anaplastic carcinomas, and other types of sarcomas were negative.

Since UEA1 stains blood vessels of both normal and tumour tissues withequal intensity it is not an obvious tumour vasculature-specifictargeting agent. However we observe that UEA1 staining has potentialapplication in studying distribution of vessels in relation to variousnormal and pathological events. Since blood vessel invasion is one ofthe most important diagnostic and prognostic parameters used bypathologists in the evaluation of neoplastic conditions, UEA1 andmimetics thereof such as peptides, peptidomimetics and/or small organicmolecules which mimic UEA1 have value in establishing the diagnosis oflymphovascular involvement.

Thus, the use of UEA1 and its mimetics as disclosed herein in thediagnosis/prognosis of conditions by observation of vascular involvementand corresponding compositions which may be adapted for imaging as inthe first aspect of the above is a further aspect of the invention.

SUMMARY OF THE INVENTION

In one general aspect, the invention is a pharmaceutical formulationcomprising a pharmaceutical agent and a bioadhesive ligand, saidbioadhesive ligand comprising an organocyclic (C,N, O and/or S) moiety,said organocyclic moiety a polyhydroxy- or polyalkoxy-substituted moiety(at least 2 hydroxy or 2 alkoxy groups, respectively.)Polyhydroxy-substituted organocyclic moieties are preferred. Forpolyalkoxy-substituted organocyclic moieties, C₁-C₅ alkoxys arepreferred, and C₁-C₃ alkoxys are more preferred, where for example a C₂alkoxy is ethoxy.

The ligand may be bound, either covalently or noncovalently, to acarrier entity comprising the pharmaceutical agent. The ligand ispreferably bound to the surface of the carrier.

In embodiments of particular interest, the carrier entity is selectedfrom the group consisting of a nanoparticle, a microparticle, and aliposome.

In some preferred embodiments, the backbone of the organocyclic moietycomprises a backbone ring that consists of 5 to 7 atoms. (For purposesherein, benzene has a backbone of 6 carbon atoms in a single ring,naphthalene a backbone of 10 carbon atoms and has two backbone rings,each consisting of 6 carbon atoms, the two rings sharing two carbonatoms). The backbone ring of 5 to 7 atoms may be unsaturated (i.e.,aromatic). In some highly preferred embodiments, all the atoms of thering backbone are carbon atoms.

Preferred backbones for the organocyclic moiety are those identical tothat of a radical selected from the group consisting of phenyl, napthyl,cyclohexyl, benzyl, benzoyl, pyridine and dihydrobenzopyran. It isparticularly preferred that such a backbone is substituted with 2 ormore hydroxy radicals, most preferably 2 to 4 hydroxy radicals. Highlypreferred organocylcic moieties are galloyl or trimethoxyphenylradicals.

In a highly preferred set of embodiments, the organocyclic moiety iscovalently linked to a scaffold moiety. In one such embodiment, thebioadhesive ligand comprises two or more organocyclic moieties linked bya scaffold moiety. Among the preferred constructs are those wherein theshortest ring-to-ring length along the scaffold and between the twoorganocyclic moieties is from 1 to 20 atoms. (To illustrate, in compound2, described below, the shortest ring-to-ring length between the napthyland chloro-phenyl radicals is 6, between the napthyl and biphenylradicals it is 5.)

A preferred group of scaffold moieties comprises a moiety selected fromthe group consisting of amino acids, guanidines, hydantoins,thiohydantoins, thioureas, cathechins, acylamines, dicyclicamines,tricyclicamines, and saccharides. A scaffold comprising an amino acid ishighly preferred, as are ones comprising a peptide of at least 1 aminoacids (preferably 2 to 50, more preferably 2 to 20, most preferably 2 to6 amino acids). It is also highly preferred that a trihydroxybenzoyl ortrimethoxybenzyl moiety be linked (either directly or via a linker) tothe amino acid or amino acids through the amide functionality. Lysine isa highly preferred amino acid for purposes of building the scaffold.

Preferably X comprises 2 to 10 organocylic moieties, each linked to thelinear backbone either directly or by a linker moiety backbone that doesnot exceed 10 atoms.

Another preferred scaffold moiety is an acylamine. Preferred acylaminesare those of the structure X—NH—(C═O)—Y, where X comprises a linearbackbone comprising at least two atoms (preferably 2 to 20) selectedfrom the group, C and N. More preferably X comprises 2 to 10 organocylicmoieties, each linked to the linear backbone either directly or by alinker moiety backbone that does not exceed 10 atoms.

Y preferably comprises a polyhydroxy or polymethoxy organocyclic moiety.It is particularly preferred that such an organocylic moiety is linkedto the (C═O) group of the acylamine either directly or by a linkermoiety backbone that does not exceed 10 atoms. In a preferred set ofembodiments, the Y moiety is selected from the group consisting of3,4,5-trihydroxyphenyl, 3,4,5-trimethoxyphenyl, 4-biphenylmethyl, and4-ethyl-4-biphenylmethyl. In one highly preferred embodiment, —(C═O)—Yis a galloyl group.

In one set of preferred embodiments, the R group (e.g., thecyclohexylmethyl moiety of cyclohexylalanine) of at least one amino acidis linked to the X moiety of the acylamine, said amino acid selectedfrom the group consisting of D-Norleucine, L-norleucine, D-tyrosine,L-tyrosine, D-cyclohexylalanine, L-cyclohexylalanine, D-arginine, andL-arginine. An organocyclic moiety can be linked to the X moiety of theacyl amine, said organocyclic can for example be selected from the groupconsisting of D-napthylmethyl, L-napthylmethyl, and L-p-chloro-benzyl.

As regards examples of specific bioadhesive ligands, the bioadhesiveligand comprises a compound selected from the group consisting of thosecompounds specified in Tables 1-6, 7A, 7B, and 8, below. (The foregoingtakes into account, for example, that the R1, R2, and R3 compounds inthe individual columns in Tables 1, 2, and 3, were the compounds used togenerate the somewhat smaller R1, R2, and R3 moieties in the chemicaldiagram (equivalent to —NH—CH2-CHR1-N(−)—CH2-CHR2-NH—CO—R3) immediatelypreceding those tables). As a result, the appearance of Nap-ala fornapthylalanine as an R1 in Table 1 indicates that the corresponding R1radical in the chemical diagram is napthylmethyl- and the appearance of3,4,5-trimethoxybenzoic acid as an R3 in Table 1 indicates that thecorresponding R3 radical in the chemical diagram is 3,4,5,trimethoxyphenyl. An analogous situation will be seen to exist for otherTables)

As to Table 4, it is preferred that the ligand comprises a compound thathas a Single Tier Assay Avg % inhibition at 250.0 (μg/ml) of at least30, more preferably at least 20.

As to Table 6, it is preferred that the ligand comprises a compound thathas a Single Tier Assay IC50(50 ug/ml) less than 250, preferably lessthan 100, most preferably less than 30.

As to Table 7(a) it is preferred that the bioadhesive ligand comprises a2-copy structure specified in Table 7(A) that has a 2nd Tier IC50 value(uM) of 350uM or less, preferably less than 200 uM, more preferably lessthan 100 uM.

As to Table 7(b) it is preferred that the bioadhesive ligand comprises acompound that is a 4-copy structure specified as having a 2nd Tier IC50value (uM) of 250 uM or less, more preferably less than 200 uM, evenpreferably less than 50 uM, most preferably less than 3 uM.

As to Table 8, it is preferred that the bioadhesive ligand comprises acompound that has an IC 50 (uM), in a 2nd tier assay, that is less than150, preferably less than 15.

The tested compounds described in Tables 1, 2, 3, 4, 5, 6, 7, 7A, 7B,and 8 are themselves aspects of the invention.

In another general aspect, the invention is a method of administering apharmaceutical formulation to an organism having an intestinalepithelium (preferably a mammal, most preferably a human), said methodcomprising administering a pharmaceutical formulation of Claim 1. In oneset of embodiments of interest, the bioadhesive ligand is covalently ornoncovalently bound (preferably on the surface) to a carrier comprisingthe pharmaceutical agent.

The present inventors have carried out very extensive investigationswith a view to identifying, testing and preparing alternative substancesshowing an affinity for epithelial cells, and hence a “bioadhesive”capacity making them useful as moieties, ingredients or coatings inenterally-administered pharmaceutical formulations.

We screened a number of combinatorial libraries, including both peptideand non-peptide organic molecules, in competitive assays with the lectinUEA-1, a lectin protein of approximately 60 kDa derived from furze (Ulexeuropaeus) that is known to bind to fucose residues and in particular isknown to bind to epithelial cells.

What we have found is that cyclic organic groups having two or more andpreferably three or more hydroxy or hydroxy-bearing substituents can bebinding-active moieties with respect to epithelial cells includingepithelial cells of the intestinal tract. Organic compounds having suchbinding-active moieties, and particularly having two or more of them onan organic skeleton or “scaffold”, can be used as bioadhesive ligands inpharmaceutical formulations.

The cyclic group may be carbocyclic or heterocyclic. It may be aromatic,non-aromatic, fused aromatic or fused partly-aromatic ring systems.

Polyhydroxy-substituted aromatic groups are preferred, e.g. diols,triols, tetrols etc of phenyl and related aryl ring systems e.g.naphthyl, or also alicyclics such as cyclohexenyl. The phenyl or relatedring may be joined to a molecular skeleton or scaffold as a benzyl orbenzoyl group, or the equivalent for the related ring systems.

Hydroxy groups on the ring may be vicinal.

In particular we have found good results with trihydroxyphenyl groups,which may be linked to a scaffold as trihydroxybenzyl or benzoyl.

The most preferred binding-active moiety that we have found is based ona 3,4,5-hydroxyphenyl group which may be joined to a scaffold e.g. viaan amide or other acyl link, so that it constitutes a galloyl(3,4,5-hydroxybenzoyl) group.

Preferably the hydroxy groups take the —OH form, although thiolanalogues and masked e.g. alkoxylated forms may also be useful.

As referred to above, we have found that good results are achieved whentwo or more, and preferably three or more, binding-active moieties asspecified above are provided on an organomolecular scaffold or skeleton.A wide variety of options exist for this scaffold but of course it ispreferably biologically compatible in the sense that it will not breakdown to harmful substances, and generally preferably contains nothingother than carbon, nitrogen, oxygen, sulphur and hydrogen. It may belinear, branched, cyclic or any combination of these.

Preferably the scaffold consists of hydrocarbon entities linked viafunctional groups. Suitable functional groups are preferably selectedfrom but not limited to amino, amido, acyl, ether, ester, carboxylicacid and urea linkages.

In view of their established biological acceptability, molecularscaffolds based on or comprising amino acid units, and/or analogues orderivatives thereof, are preferred. The scaffold may be or comprise anamino acid, peptide, oligopeptide (preferably from 2 to 10 and morepreferably from 1 to 6 amino acids) substituted with one or more andpreferably plural of the binding-active moieties mentioned above.Natural or synthetic amino acids may be used in the scaffold.

Non-peptide scaffolds are also possible. The skilled person is alreadyaware of peptidomimetic molecules and molecular frameworks ofestablished effectiveness, and these include, among various types ofmolecules using the functional groups and linkages mentioned above,molecules comprising heterocyclic rings, guanidines, hydantoins,thiohydantoins, thioureas, catechins, acylamines, saccharides and soforth.

Where the scaffold comprises an amino acid, peptide, oligopeptide oranalogue thereof at least one binding-active moiety may be linked at theC-terminal of the scaffold.

The scaffold may provide a linear or cyclic backbone from which thebinding-active groups are branched, optionally via branch spacer chainssuch as hydrocarbon chains.

Links between the binding-active moieties and the scaffold may be viaamino, amido, acyl, ether, alkylene, alkenylene or other suitablefunctionalities, or any combination of these.

While many of the binding-active compounds (ligands) proposed herein arebelieved to be novel, it is also possible to use existing compounds andanalogues thereof such as tannic acid and the other tannins, which ingeneral feature plural galloyl substituents on a sugar substrate. Forthese known substances, this is a newly-proposed use and formulation.

Considering the ligand compound as a whole (one or more binding-activemoieties plus any scaffold) or multimers thereof its molecule isdesigned in line with conventional biochemical practice so as to besufficiently stable in the enteric tract. By comparison with the lectinsas previously discussed, the molecular weight of the ligand may be lowand this, together with a suitable chemical make up, can providestability, reduction in the potential for immunogenicity andcytotoxicity, as well as facilitating the manufacture and processing ofsynthetic ligands. A preferred molecular weight is less than 5000,preferably less than 2000 or 1500, but does not exclude multimersthereof.

However it should be noted that the present proposals also comprehendthe possibility of providing the binding-active moieties whoseeffectiveness has been disclosed here on other types of molecule. Forexample they may be provided as substituents or grafts on a polymericexcipient used in the pharmaceutical formulation, such as abiodegradable polymer. The ligand molecule as a whole may be covalentlyor non-covalently bound on or into the pharmaceutical formulation.Similarly, the ligand molecule as a whole may be covalently ornon-covalently bounds to a drug or antigen or adjuvant.

The novel pharmaceutical formulations exploiting these binding-activemoieties and ligand compounds are one aspect of the invention. The useof the binding-active moieties and ligand compounds to enhance drugdelivery in an enteric e.g. oral formulation is another aspect.

The ligand compounds proposed herein are for the most part novel, and inthemselves, as UEA1 mimics, are an aspect of the invention claimed here.

The corresponding methods are also aspects of the invention claimedhere, namely methods comprising the synthesis of the novel bindingcompounds, and methods of preparing pharmaceutical formulationscomprising incorporating the ligand compounds—whether novel or not—intothe formulation by blending, binding, coating or by other means.

In particular embodiments of the invention, one of the aforementionedbioadhesive ligands is covalently or non-covalently bound to a carrierentity comprising a pharmaceutical agent. For example, the carrierentity is selected from the group consisting of a nanoparticle,microparticle and liposome. It is preferred that the carrier entity havea largest dimension that is in the range of 10 nm to 500 μm, asdiscussed in more detail elsewhere herein. In particular embodiments ofthe invention, the pharmaceutical agent is a drug or therapeutic agent.In other specific embodiments, the pharmaceutical agent is a pathogenantigen.

Certain aspects of the invention involve the use of the bioadhesiveligands to target delivery of pharmaceutical agents.

In one aspect, the invention is a method of administering apharmaceutical agent to an organism having intestinal epithelium, saidmethod comprising contacting said intestinal epithelium with one of theaforementioned bioadhesive ligands that is covalently, or non-covalentlybound to, a carrier entity. In preferred the embodiments, the organismis a mammal. Most preferably, the mammal is a human.

In particular embodiments of the method, the carrier entity is from thegroup consisting of a nanoparticle, microparticle or liposome.Preferably, the carrier entity has its major dimension in the range of10 nm to 500 μm. In preferred embodiments, the carrier entitydrug-loaded or drug-encapsulated. The preferred route of administrationfor delivery of the ligand-carrier entity is the oral route. Otherpossible routes are the rectal, subcutaneous, intramuscular andintravenous routes.

As used herein, the term “carrier entity” is defined as a particle ordroplet that can carry a pharmaceutical agent. A microparticle isdefined as a particle whose major dimension in the range 1 to 5 μm, mostpreferably in the range 1 to 3 μm. A nanoparticle is defined as aparticle whose major dimension is less than 1μ, preferably in the range1 nm to 500 nm, most preferably in the range 10 nm to 500 nm.

As used herein, the major dimension of a spherical particle is itsdiameter, or a rod shaped particle, its length. For other particles, itis the longest dimension possible for the particle.

Nano- and microparticles that are loaded with, or encapsulate,pharmaceutical agents, can be coated with the bioadhesive ligands, suchas those of the present invention, that target intestinal epitheliumtissue. The coating can be effected by covalent or non-covalent bonding.The covalent bonding can be achieved by adsorption or any other coatingprocess. In either case, the bonding can be made to completed particlesor to particle components that subsequently form part of the particles.

Biodegradable Particles are Preferred.

Pharmaceutical agents can, in the alternative, be directly linked abioadhesive ligand.

A “pharmaceutical agent” is a therapeutic or diagnostic agent.Therapeutic agents are those that are administered either to treat anexisting disease or prophylactically to protect against a potentialfuture disease. Diagnostic agents are any agents that are administeredas part of a diagnostic procedure.

Examples of therapeutic agents are drugs, genes, gene-delivery vectors,and antigens/vaccines.

Drugs include, for example, analgesics, anti-migraine agents,anti-coagulant agents, anti-emetic agents, cardiovascular agents,anti-hypertensive agents, narcotic antagonists, chelating agents,anti-anginal agents, chemotherapy agents, sedatives, anti-neoplastics,prostaglandins and antidiuretic agents, antisense oligonucleotides,gene-correcting hybrid oligonucleotides, ribozymes, RNA interference(RNAi) oligonucleotides, silencing RNA (siRNA) oligonucleotides,aptameric oligonucleotides and triple-helix forming oligonucleotides,DNA vaccines, adjuvants, recombinant viruses.

Examples of gene-delivery vectors are DNA molecules, viral vectors (E.g.adenovirus, adeono-associated virus, retroviruses, herpes simplex virus,and sindbus virus), and cationic lipid-coated DNA and DNA-dendrimers.

Drugs include conventional small molecule drugs, proteins,oligopeptides, peptides, and glycoproteins.

Examples of drugs are as insulin, calcitonin, calcitonin gene regulatingprotein, atrial natriuretic protein, colony stimulating factor,betaseron, erythropoietin (EPO), interferons (E.g. α, β or γinterferon), somatropin, somatotropin, somatostatin, insulin-like growthfactor (somatomedins), luteinizing hormone releasing hormone (LHRH),tissue plasminogen activator (TPA), growth hormone releasing hormone(GHRH), oxytocin, estradiol, growth hormones, leuprolide acetate, factorVIII and interleukins (E.g. interleukin-2). Representative drugs alsoinclude: analgesics (E.g. fentanyl, sufentanil, butorphanol,buprenorphine, levorphanol, morphine, hydromorphone, hydrocodone,oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen andpaverin); anti-migraine agents (E.g. sumatriptan and ergot alkaloids);anti-coagulant agents (E.g. heparin and hirudin); anti-emetic agents(E.g. scopolamine, ondansetron, domperidone and metoclopramide);cardiovascular agents, anti-hypertensive agents and vasodilators (E.g.diltizem, clonidine, nifedipine, verapamil, isosorbide-5-mononitrate,organic nitrates and agents used in treatment of heart disorders);sedatives (E.g. benzodiazepines and phenothiozines); narcoticantagonists (E.g. naltrexone and naloxone); chelating agents (E.g.deferoxamine); anti-diuretic agents (E.g. desmopressin and vasopressin);anti-anginal agents (E.g. nitroglycerine); anti-neoplastics (E.g.5-fluorouracil and bleomycin); prostaglandins; and chemotherapy agents(E.g. vincristine).

Examples of antigens that are therapeutic agents are tumor antigens,pathogen antigens and allergen antigens. A vaccine preparation willcontain at least one antigen. “Pathogen antigens” are thosecharacteristic of pathogens, such as antigens derived from viruses,bacteria, parasites or fungi.

Examples of important pathogens include vibrio choleras, enterotoxigenicE. Coli, rotavirus, Clostridium difficile, Shigella species, Salmonellatyphi, parainfluenza virus, influenza virus, Streptococcus mutans,Plasmodium falciparum, Staphylococcus aureus, rabies virus andEpstein-Barr virus.

Viruses in general include the following families: picronaviridae;caliciviridae, togaviridae; flaviviridae; coronaviridae; rhabodviridae;filoviridae; paramyxoviridae; orthomyxoviridae; bunyaviridae;arenaviridae; reoviridae; retroviridae; hepadnaviridae; parvoviridae;papovaviridae; adenoviridae; herpesviridae and poxyviridae.

Bacteria in general include but are not limited to: P. aeruginosa; E.coli; Klebsiella sp.; Serratia sp; Pseudomanas sp.; P. cepacia;Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumonias; S. aureus;Haemophilus sp.; Neisseria sp.; N. meningitidis; Bacterodies sp.;Citrobacter sp.; Branhamella sp.; Salmonelia sp.; Shigella sp.; S.Lesteria sp., Pasteurella multocida; Streptobacillus sp.; S. pyogenes;Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Spirillum sp.;Fusospirocheta sp.; Treponema pallidum; Borrelia sp.; Actinomycetes;Mycoplasma sp.; Chlamydia sp.; Rickettsia sp., Spirchaeta; Legionellasp.; Mycobacteria sp.; Urealplasma sp.; Streptomyces sp.; Trichomorassp.; and P. mirabilis

Parasites include but are not limited to: Plasmodium falciparum, P.vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana, L.tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma mansoni, S. haematobium, S. japonium; Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobus vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginitis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocytis carinii, Babesia bovis, B. divergens, B. microti,Isospore belli, L. hominis; Dientamoeba fragiles; Onchocerca volvulus;Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongylyscantonensis; Hymenolepis nan; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westermani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica, Sarcoptes scabiei, Pediculus humanus; Phtirius pubis; andDermatobia hominis.

Fungi in general include but are not limited to: Crytpococcusneoformans; Blastomyces dematitidis; Aiellomyces dermatitidisHistoplasfrai capsulatum; Coccidiodes immitis; Candids species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, Aspergillus species, including A. fumigatus, A. flavusand A. niger, Rhizopus species; Rhizomucor species; Cunnighammellaspecies; Apophysomyces species, including A. saksenaea, A. mucor and A.absidia; Sporothrix schenckii, Paracoccidioides brasiliensis;Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.

Antigens that are allergens can be haptens, or antigens derived frompollens, dust, molds, spores, dander, insects and foods. Specificexamples include the urusiols of Toxicodendron species and thesesquiterpenoid lactones.

Examples of adjuvants: Freund's Complete Adjuvant, Freund's IncompleteAdjuvant, Hunter's Titermax, Gerbu Adjuvant, Ribi's Adjuvant, MontanideISA Adjuvant, Aluminum Salt Adjuvants and Nitrocellulose adsorbedprotein.

In another general aspect we have confirmed the effectiveness of UEA1and of its mimics in binding to human intestinal tissue sections. Inview of the relationships noted above between various disease states, weput forward the first aspect of the present invention which is methodsand compositions for assessing the status of GIT cells by means ofimaging, using UEA1 or a UEA1 mimetic as a localisation agent whichbinds characteristically to epithelial cells. Relevant disease statesinclude any of those mentioned above, for example, colon carcinoma,ulcerative colitis and Crohn's Disease. The UEA1 or UEA1 mimeticlocalisation agent may be exploited for diagnostic/prognostic imaging inany of a variety of ways and these may in themselves be conventional.For example the UEA1 and the UEA1 mimetic may be used in an immunoassayprocedure with an antibody therefor or other specific binding substance,and an imaging agent or agents (e.g. a colour staining test kit) forproviding a characteristic image/colour when reacted with the antibodyor other specific binding substance.

Alternatively UEA1 or UEA1 mimetic may be directly labelled, e.g.biotinylated or by some other means, so that its bound presence on thetest cells can be verified by reaction with avidin or the appropriateother imaging substance(s) or test for the type of label used. Otherpossibilities include NMR imaging and radiolabelling.

Compositions for the present purpose may comprise the UEA1 or UEA1mimetics, which may be labelled, as part of a diagnostic/prognosticimaging kit including any necessary complementary binding substances andimaging media.

The use of UEA1 and its mimetics as disclosed herein in thediagnosis/prognosis of conditions by observation of vascular involvementand corresponding compositions which may be adapted for imaging as inthe first aspect of the above is a further aspect of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Synthesis of compounds having four copies of gallic acid.

FIG. 2 Surface binding and uptake of MSI35-2 gallic acid mimetic coatedparticles.

FIG. 3 Surface binding and uptake of UEA1 coated particles.

FIG. 4 Surface binding & uptake of biocytin mimetic coated particles.

FIG. 5 MSI 35: Biotinylated 2-copy and 4-copy structures.

FIGS. 6 and 7. Stained human tissue samples.

FIG. 8 Endothelial cell permeability assay.

DETAILED DESCRIPTION

There now follows a detailed description of those aspects of ourexperimental work leading to the identification of the particularbinding-active moieties disclosed herein, ligand compounds bearing them,verification of their activity and various examples of ligand compoundsembodying the invention.

The search for small molecular weight ligands capable of binding surfacereceptors of epithelial cells began with the screening of variouscombinatorial libraries, which contained both peptides and non-peptide(organic) molecules. These libraries were synthesized in positionalscanning format in which oligopeptide mixture sets comprise onepredetermined residue at a single predetermined position of theoligopeptide chain (Pinilla et al. U.S. Pat. No. 5,556,762, Pinilla etal. 1992. BioTechniques. Vol 13, No 6).

The epithelial cell binding ability of compounds and mixtures obtainedfrom synthetic combinatorial libraries was determined by competitionassays. These assays were set up to measure the inhibition of binding ofbiotinylated UEA-1 to membrane preparations of the epithelial cell lineCaco-2 (colon carcinoma cells-2), by compounds and mixtures of thecombinatorial libraries. This cell line is a conventional model forepithelial cells. The ability of a compound or compounds to inhibitUEA-1 binding would suggest that this chemical itself was binding tofucose residues on the surface of epithelial cells and was hence apotential ligand.

Two competition inhibition assays were used in this case, namely singletier and two tier assays. These assays differed from each other in thatin the single tier assay, the mixtures/compounds and the biotinylatedUEA-1 were incubated together with the Caco-2 cell membranepreparations, while in case of the two-tier assay, the mixture/compoundswere allowed to incubate alone with the cell membranes in the absence ofbiotinylated UEA-1, which was added in the next step. Addition of theextra step in the two-tier assay was to ensure that the compoundsinhibiting the binding of biotinylated UEA-1 to the Caco-2 cellmembranes were doing so by themselves binding to surface receptors onthe Caco-2 cells and not to the biotinylated UEA-1. Each assay will bedescribed in detail.

Preparation of Caco-2 Cell Membrane (P100) and Cytosolic (S100)Fractions:

-   1. Confluent Caco-2 cell monolayers (grown in 75 cm² flasks for up    to 1 week at 37° C. and 5% CO₂) were washed twice in Dulbecco's PBS    (DPBS).-   2. Cell monolayers were treated with 10 mM EDTA-DPBS for 5-10 min at    37° C. and cells were harvested by centrifugation at 1000 rpm for 5    min.-   3. Cells were washed 3 times in DPBS.-   4. The cell pellet was resuspended in 3 volumes of ice cold HED    buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1    mM phenylmethylsulphonyl fluoride (PMSF)) and the cells were allowed    to swell for 5 min on ice.-   5. The cells were homogenised for 30 sec.-   6. The homogenates were centrifuged in hard walled tubes at 40,000    rpm for 45 min at 4° C.-   7. The supernatant (S100) was removed and the pellet (P100) was    resuspended in HEDG buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM    dithiothreitol, 100 mM NaCl, 10% glycerol, 1 mM PMSF): 3 volumes of    buffer were added, the pellet was resuspended and centrifuged at    1000 rpm for 2 min. The supernatant was removed and stored on ice.    The procedure was repeated adding the second supernatant to the    first.-   8. The protein concentration was determined using e.g. a Bio-Rad    protein assay.-   9. All fractions were stored at −80° C. prior to use.    Single Tier Assays:-   1. 96-well microtiter plates were coated with membrane preparations    of Caco-2 cells either by allowing them to incubate on the plates    for 2½ hours at room temperature or by incubating them overnight at    4° C. 100 μl of 10 μg/ml (in 0.05 M carbonate buffer, pH 9.6) of    membrane preparation was added to each well.-   2. The plates were flicked out, patted dry and blocked with bovine    serum albumin (BSA)/DPBS (200 μl/well) for 1 to 4 hours at room    temperature after which they were washed three times in water.-   3. 50 μl/well (in 1.5% BSA/DPBS) of the mixtures/compounds from the    combinatorial libraries were added to the wells. Control wells were    also set up in which the Caco-2 cell membrane preparations were    incubated with unconjugated UEA-1. A 1 in 4 dilution series of this    control compound was set up starting from 0.04 μg/ml to 160 μg/ml    (50 μl/well in 1.5% BSA/DPBS).-   4. Biotinylated UEA-1 at a final concentration of 1 μg/ml (50 μl in    1.5% BSA/DPBS) was added to each well and the plates were left to    incubate overnight at 4° C.-   5. Following overnight incubation the plates were washed thoroughly    (3-6 times). The plates were flicked out, patted dry and    biotinylated UEA-1 was detected with commercial Streptavidin    conjugated to horseradish peroxidase (HRP) (CalBiochem). This    reagent was added to each well at a 1:5000 dilution prepared in 1.5%    BSA-DPBS such that 100 μl of the reagent was added to each well to    achieve the final concentration. The plates were left to incubate    for an hour at room temperature.-   6. The plates were then washed 3-6 times and the biotin-streptavidin    binding was detected by adding an HRP substrate OPD (orthophenyl    diamine) to each well. 100 μl/well of this substrate at a final    concentration of 1.6 mg/ml was added to each well. Before adding to    the wells, this substrate was activated by addition of 50 μl of 3%    H₂O₂ per plate and the reaction was allowed to develop in the dark.-   7. The reaction was stopped by adding 50 μl of 4 N H₂SO₄ to each    well after approximately 5-10 minutes or when sufficient colour had    developed. The OD₄₉₀ (absorbance at 490 nm) of each well was    measured using a conventional 96 well plate reading    spectrophotometer.    Two Tier Assays:-   1. 96 well plates were coated with Caco-2 cell membrane preparations    in the same manner as described in the single tier assay.-   2. Plates were flicked out, patted dry and blocked with 1.5%    BSA-DPBS (200 μl/well) for 1-4 hours at room temperature and then    washed three times in water.-   3. The compounds/mixtures from various combinatorial libraries were    then added to individual wells (100 μl/well in 1.5% BSA-DPBS).    Control wells were also set up containing a range of concentrations    of purified UEA-1 which were set up as 1 in 4 dilutions starting    from 0.04 μg/ml to 160 μg/ml. 100 μl at these final concentrations    were added to each well.-   4. The plates were then left to incubate overnight, a step that    would allow the mixtures and compounds to interact with the Caco-2    cell membranes without the presence of the competitor (biotinylated    UEA-1) itself.-   5. Following overnight incubation, the plates were washed thoroughly    in water 3-6 times.-   6. 100 μl/well of biotinylated UEA-1 at a final concentration of 1    μg/ml in 1.5% BSA-DPBS was added to each well and the plates were    allowed to incubate for two hours at room temperature.-   7. After washing the plates 3-6 times, biotin was detected by use of    commercial Streptavidin conjugated to HRP, which was added to each    well at a 1:5000 dilution prepared in 1.5% BSA/DPBS such that 100 μl    of the reagent was added to each well to achieve the final    concentration. The plates were left at room temperature for an hour.-   8. After 3-6 washes, 100 μl/well of OPD (final concentration 1.6    mg/ml) activated by H₂O₂ was added to each well and the reaction was    allowed to develop in the dark.-   9. The reaction was stopped by adding 50 μl of 4 N H₂SO₄ after    approximately 10-15 minutes or when sufficient colour had developed.-   10. The absorbance at 490 nm of each plate was measured by    spectrophotometry.    Results:

The results are illustrated as the percentage inhibitory activity or theIC₅₀ (the concentration of the compound at which 50% inhibition of UEA-1was reported). The absorbance at 490 nm of the unconjugated UEA-1controls (1:4 dilutions: 160 μg/ml to 0.04 μg/ml) was used to set up astandard curve. The highest concentration of unconjugated UEA-1 was 160μg/ml and wells containing this level of protein showed little or nocolour change, as high levels of previously incubated UEA-1 bound to themajority of UEA-1 binding sites on the Caco-2 membrane preparations,thereby leaving no sites for biotinylated UEA-1 (added later) to bind toand hence no biotinylated UEA-1 was detected.

Wells containing 0.04 μg/ml of unconjugated UEA-1 showed high absorbanceat 490 nm as low concentration of UEA-1 meant that most UEA-1 receptorswere left unbound, which allowed biotinylated UEA-1 to bind to thesereceptors on the Caco-2 cell membranes, thus resulting in highabsorbance of these wells at 490 nm. The absorbance at 160 μg/ml wastaken as 100% inhibition and the absorbance at the other end of thescale (0.01 μg/ml) was taken as 0% inhibition. The percentage inhibitionof each compound or mixtures of compounds was estimated from similarbinding curves. The IC₅₀ values of the active compounds was determinedusing serial dilutions of each compound.

Using combinatorial chemistry, a large number of diverse chemicalcompounds, both peptide and non-peptide (organic) referred to aslibraries were tested in single and two tier assays. Each librarycomprises a common scaffold or framework. Compounds of each library aresynthesized by arranging a wide range of side chains and groups bothbranched and linear in different sequences on the scaffold backbone. Ina different library the same elements can be arranged in a similarmanner or in a different manner on another type of scaffold (forreference see Meyer et al., U.S. Pat. No. 5,859,190, Houghten, U.S. Pat.No. 4,631,211, Pinilla, U.S. Pat. No. 5,556,762). By way of example,some of the organic backbones used in combinatorial chemistry are listedbelow. The libraries screened in order to identify the active compoundsof the invention were not limited to the backbone structures definedbelow.

Amongst the wide range of libraries tested, mixtures from thiohydantoinbased, N-6-acylamino bicycylic guanidine based, N-acylamine based andpolyphenylurea based libraries showed high inhibitory activity. Tonarrow the search, individual compounds from these active mixtures weredeconvoluted and each tested for inhibitory activity. Results revealedthat individual purified compounds from most libraries showed inhibitoryactivity. These included compounds from the N-acylamine-based libraries.A structure of such acylamines is e.g. as represented by formula A.

The acylamines were synthesized on solid phase resin. Here R1 and R2groups were derived from amino acids that were coupled usingconventional tBOC chemistry which involves blocking the N terminus ofeach incoming amino acid by BOC (N-tertbutoxyl carbonyl) to avoid itsparticipation in the reaction. The N terminus is unblocked once theamino acid is attached. In case of synthesis of acylamines, the amidebonds of the amino acids were first methylated and then reduced toamines. The N-terminus of the developing chain was acylated with acarboxylic acid adding R3. The amine seen on the left side of themolecule was derived from the solid phase mBHA resin which has an aminogroup extending out that reacts with the carboxy terminus of theincoming amino acid. Cleavage of this amine from the solid supportduring incubation with hydrogen fluoride results in the release of thisamino group thus forming the amino terminus of the acylamine.

The scheme is shown below.

Tables 1, 2 and 3 show the inhibitory activity of compounds belonging tothree N-acylamine-based libraries. TABLE 1 50 μg/ml 250 μg/ml 50 μg/ml250 μg/ml Av % Av % Av % Av % R1 R2 R3 Inhibition Inhibition InhibitionInhibition  5. Nap-ala Nap-Ala 3,4,5-Trimethoxybenzoic Acid 16 54 39 51 8. Nap-ala Nap-ala 4-Biphenylacetic Acid 59 85 0 50 10. Nap-ala Nap-ala3,4,5-Trimethoxybenzoic Acid −65 41 24 66 20. Nap-Ala Nap-ala3,4,5-Trimethoxybenzoic Acid −121 24 52 75 23. pCl-F Nap-Ala4-Biphenylacetic Acid 52 21 39 59 25. pCl-F Nap-Ala3,4,5-Trimethoxybenzoic Acid −173 66 65 63 26. pCl-F Nap-ala4-Ethyl-4-Biphenylcarboxylic Acid −116 −2 39 56Av % Inhibition:—Average Percent Inhibition

TABLE 2 Two Tier Assay N-acyl triamine Library TP1012 50 ug/ml 250 ug/mlR1 R2 R3 Avg % Inhib Avg % Inhib 86 D-Nle D-chAla Gallic acid 61.6130876.22587 125 L-Leu L-Phe Gallic acid 61.02335 77.78063 87 D-NleD-Arg(Tsl) Gallic acid 55.81319 65.64472 84 L-Tyr(Brz) L-chAla Gallicacid 54.9682 70.64093 92 L-Nle D-chAla Gallic acid 54.8899 67.69989 90D-Nle L-chAla Gallic acid 53.65788 69.94 91 L-Nle L-Tyr(Brz) Gallic acid53.05769 65.97437 94 L-Nle L-Arg(Tsl) Gallic acid 51.51322 66.36993 78D-Tyr L-chAla Gallic acid 47.10349 61.0039 93 L-Nle D-Arg(Tsl) Gallicacid 46.83358 58.5174 85 D-Nle L-Tyr(Brz) Gallic acid 46.51721 66.2115296 L-Nle L-chAla Gallic acid 42.93428 65.78116 74 D-Tyr D-chAla Gallicacid 42.91734 57.91947 144 L-pF-Phe L-pF-Phea,a,a-(Trifluoro-m-Tolyl)acetic acid 42.26244 66.71332 89 D-Nle D-TyrGallic acid 42.12294 65.78345 95 L-Nle D-Tyr Gallic acid 41.9864466.4157 141 L-pF-Phe L-pF-Phe 3,4 DichloroPhenylacetic acid 41.4319172.63565 75 D-Tyr D-Arg(Tsl) Gallic acid 40.14659 51.4412 88 D-NleL-Arg(Tsl) Gallic acid 38.0323 59.11027 82 L-Tyr(Brz) L-Arg(Tsl) Gallicacid 37.9772 55.92339Av % Inhibition:—Average Percent Inhibition

TABLE 3 Single Tier Two Tier Acylamine Library MSI 22 Assay Assay R1 R2R3 62.5 μg/ml 250 μg/ml 62.5 μg/ml 250 μg/ml L-NapAla L-pCl- Gallic Acid31.5 46.9 26.7 54.7 Phe D-Nve D-chAla Gallic Acid 22.0 47.8 20.6 48.5D-Tyr(Et) D- Gallic Acid 27.2 48 30 61 Arg(Tos) D-Tyr(Et) L-chAla GallicAcid 8.5 33.3 31.8 52.7 D-Nve D-Val Gallic Acid 13.7 46 12.1 29.8 D-Tyr-L-chAla 3,4,5-Trimethoxy benzoic −0 −11.7 39.7 58.9 (BrZ) Acid D-NapalaL-pCl- Gallic Acid −14.7 7.4 23.4 43.3 Phe D-Napala D-Val Gallic Acid4.7 26.8 24.2 46.9 L-NapAla D-Napala Gallic Acid −3 7 22.0 44.6D-Tyr(Et) L-chAla 3,4,5-Trimethoxy benzoic −9 −55.7 49.8 43.8 AcidAv % Inhibition:—Average Percent Inhibition

Compounds in these libraries have the same N-acylamine scaffold butdiffer in the arrangement of side groups on each scaffold. Library TPI1066 (Table 1) for example contains compounds with aromaticfunctionalities, library TPI 1012 (Table 2) contains compounds witharomatic and non aromatic functionalities are arranged on a N-acylaminebased scaffold, and compounds from library MSI 22 (Table 3) are acombination of the first two libraries, in that the N-acylamine scaffoldhas both aromatic groups and amino acids attached to it; results areillustrated as the average percent inhibition by 50 μg/ml or 250 μg/mlof these compounds. Note that the compounds from MSI 22 were tested atdoubling dilutions. Results are illustrated as the average percentinhibition by 62.5 μg/ml or 250 μg/ml of these compounds. Structures ofsome of the synthetic compounds tested from TPI 1066 are shown below:

From the acylamine libraries, we found that compounds bearing cyclicgroups having hydroxy or hydroxy bearing substituents at position R3were the most active inhibitors of biotinylated UEA-1 binding (Table 2).In case of the library TPI 1012, compounds with the following groups atpositions R1, R2 and R3 showed the most activity.

In the light of these results, further libraries were synthesized usinga range of carboxylic acids with the intention of assessing if multiplecopies of polyhydroxyaryl groups such as galloyl groups and some othercyclic groups increased the inhibitory activity of the compounds. Thesecompounds were synthesized on a lysine scaffold in which carboxylicacids were coupled to amines on both alpha and epsilon positions. Twodifferent constructs with each carboxylic acid were made: a ‘two copy’construct represented as formula (C) and a ‘four copy’ constructrepresented as formula(B).

A range of carboxylic acids were used to synthesize these two copy andfour copy constructs. Structures of the acids attached to lysinescaffolds of this library are given below.

By way of reference example, the reaction scheme for synthesis ofcompounds carrying four copies of gallic acid on a lysine scaffold isnow described. The typical synthesis of such compounds involved solidphase organic chemistry methodology. This reaction scheme is illustratedin FIG. 1.

-   1. A quantity of 100 mg mBHA (methylbenzhydryl amine, sub. 0.8    mmol/g) derivatized polystyrene resin was contained within a    polypropylene mesh packet called a “tea-bag”. A mesh of this nature    has previously been disclosed in Houghten, R. A., Proc Nat Acad Sci.    USA. 1985, 82, 5131. The resin functionality, methyl    benzhydrylamine, is the site of attachment for the first step in the    synthesis. The mesh packet containing the resin was contained in a    polyethylene bottle. The resin was washed three times with 5 ml    methylene chloride and neutralized three times with 5 ml of 5%    diisopropylethylamine/methylene chloride solution.    Fmoc-D-Lysine(Boc)-OH (6 eq.) was then coupled (step 1 of FIG. 1)    for two hours in the presence of HOBt (1-hydroxybenzotriazole, 6    eq.)and DIPCDI (diisopropylcarbodiimide, 6 eq.) in DMF    (dimethylformamide) to afford compound A (FIG. 1). All reagents are    utilized in six-fold excess to assure complete acylation. This    mixture was shaken with the resin for two hours. The excess reagents    were then washed out with DMF and methylene chloride.-   2. The t-Boc protecting group was removed (Step 2 of FIG. 1) with 5    ml 55% trifluoroacetic acid/methylene chloride solution for 30    minutes. The resin was then washed with methylene chloride,    isopropanol and again with methylene chloride.-   3. The Fmoc group was then removed (Step 3 of FIG. 1) with a 5 ml    solution of 20% piperidine in DMF for 30 minutes. Excess base was    removed by washing three times with DMF.-   4. The coupling procedure was repeated (Step 4 of FIG. 1) as    described above to couple Fmoc-D-Lysine(Boc)-OH to both the α and ε    amino positions producing compound B (FIG. 1).-   5. Again, the t-Boc and Fmoc groups were removed (Steps 5 and 6).-   6. The mesh packet and resin were next immersed in a solution of    gallic acid (3,4,5-trihydroxybenzoic acid, 6 eq.), HOBt (6 eq.) and    DIPCDI (6 eq.) in DMF.-   7. The reaction mixture was shaken overnight (Step 7). All couplings    were tested by the Kaiser test to verify completeness of the    reaction.-   8. Following the coupling of gallic acid, a treatment was necessary    to remove esters that formed due to the phenolic nature of gallic    acid. These esters were hydrolyzed in the final cleavage from the    resin, however they remained as an undesired side product,    complicating the post-synthesis purification process. The tea-bag    was treated with a solution of 2 ml Hydrazine in 15 ml of 10%    Methanol/90% Dioxane and shaken overnight (Step 8). The bag was    finally washed with dioxane three times.-   9. The compound was cleaved from the resin (Step 9) by hydrofluoric    acid with 5% anisole as a scavenger. This reaction was kept at 0° C.    for 90 minutes followed by a stream of nitrogen to remove excess HF.    Following extraction with 95% acetic acid/5% water and    lyophilization, the desired product C (FIG. 1) was obtained.

The biotinylated UEA-1 binding inhibitory activity of these compounds(Library N78) is shown in Table 4. TABLE 4 INHIBITORY ACTIVITY OFINDIVIDUAL COMPOUNDS CONTAINING MULTIPLE COPIES OF CARBOXYLIC ACIDS.Gallic acid D-Lys 76.1 80.4 83.1 87.7 Thioproline D-Lys Bis boc −28.2−35.5 −0.2 −29.4 3,4-(Methylenedioxy)- D-Lys Bis boc −6.2 −15.3 26.636.3 Phenylacetic Acid Rhodanine 3-acetic acid D-Lys Bis boc −6 32.5 4.816.5 Gallic Acid D-Lys Bis boc 66.6 51.3 87 91 Thioproline D-Lys Bis boc−10.9 −27.2 2.3 4.1SD:—Standard DeviationAv % Inhibition:—Average Percent Inhibition

As seen in Table 4, the polyhydroxyphenyl constructs were significantlymore active than the individual monohydroxyphenyl compounds ininhibiting biotinylated UEA-1 binding to Caco-2 cell membranes. Thenthere was the question whether or not the presence or nature of thescaffolds upon which these groups were arranged contributed to theinhibitory activity of these compounds. In order to investigate this,firstly a range (library MSI 26) of commercially available compoundshaving aromatic groups with one or more hydroxy groups, such as gallicacid, and other related compounds were tested in the inhibition assays.The structures of compounds tested in this experiment (Table 5) areshown below.

TABLE 5 Two Tier Assay Single Tier Assay Avg % Inhibition Avg %Inhibtion Library MSI 26 62.5 μg/ml 250 μg/ml 62.5 μg/ml 250 μg/mlMethyl 3,5,4- −11 −8 1 4.5 trihydroxybenzoate Lauryl gallate 20.4 47.939.1 22.5 Gallocatechin gallate 7 14.1 54.6 33.6 Epigallocatechin −12−10 10.1 0 3,4,5- 10.7 41.4 41.7 21 trihydroxybenzamide Isopropylgallate−7 8.9 10.8 3.9 Dilazep dihydrochloride −25 −25 14.7 10.4 Methylsyringate −10 0.2 17.4 8.4 4-hydroxybenzamide −1 4.2 −3 8.43,5-dihydroxybenzamide −7 −2 −1 2.4 3,5-dihydroxybenzoic −5 −7 0 1.1AcidAv % Inhibition:- Average Percent Inhibition

As seen in Table 5, the rather low (although appreciable) inhibitoryactivity of the various polyhydroxyphenyl containing compounds suggestedpreferability of substantial molecular scaffolds in binding to the UEA-1receptor. In addition, compounds with multiple polyhydroxyphenyl groupsshowed greater inhibitory activity. The results suggested the effectshould be optimised by having multiple active side groups on scaffolds.

Therefore, a range of polyhydroxy aryl groups such as galloyl and othercyclic groups, as shown below, were attached to different lysinescaffolds.

Both branched and linear skeletons as shown below were synthesized inthis library which preferably contained up to four or more hydroxylmoieties. The resulting compounds (library MSI 27) were tested in singleand two tier assays the results of which are shown in Table 6.

Analysis of the biotinylated UEA-1 binding inhibitory activity of theselinear and branched compounds shows that the activity of the compoundsincreased with the increase in the number of gallic acid groups(structures of the active compounds are shown below).

Compounds with other carboxylic acids attached as side groups on thelysine scaffolds were not as active (Table 6). TABLE 6 Inhibitoryactivity of lysine scaffolds both linear or branched with a variation ofgallic acid constructs Single Tier Two Tier Assay Assay Cpd Library MSI27 IC50 IC50 # R Comment MW (ug/ml) uM (ug/ml) uM 6 Gallic acid 2 copystructure 449.41 18.97 42.21 12.97 28.86 2 3,4-Dimethoxybenzoic acid 2copy structure 473.52 128.20 270.74 96.44 203.67 54-ethoxycarbonyl-3,5-dimethoxybenzoic acid 2 copy structure 505.52168.10 332.53 130.70 258.55 1 Syringic acid 2 copy structure 505.52250.00 494.54 250.00 494.54 8 3,5-dihydroxybenzoic acid 2 copy structure417.41 125.00 299.47 171.90 411.83 4 3,4,5-trimethoxybenzoic acid 2 copystructure 533.57 250.00 468.54 250.00 468.54 3 3,4,5-triethoxybenzoicacid 2 copy structure 617.72 250.00 404.71 250.00 404.71 74-Hydroxybenzoic acid 2 copy structure 385.41 250.00 648.66 250.00648.66 13 4-ethoxycarbonyl-3,5-dimethoxybenzoic acid 1 copystructure(N-term acetylated) 367.40 89.92 244.75 73.30 199.51 14 Gallicacid 1 copy structure(N-term acetylated) 339.35 129.70 382.20 91.52269.69 15 4-Hydroxybenzoic acid 1 copy structure(N-term acetylated)307.35 111.40 362.45 86.81 282.45 10 3,4-Dimethoxybenzoic acid 1 copystructure(N-term acetylated) 351.41 250.00 711.43 125.00 355.71 9Syringic acid 1 copy structure(N-term acetylated) 367.41 125.00 340.22101.20 275.45 16 3,5-dihydroxybenzoic acid 1 copy structure(N-termacetylated) 323.35 250.00 773.16 108.30 334.93 11 3,4,5-triethoxybenzoicacid 1 copy structure(N-term acetylated) 423.51 250.00 590.31 250.00590.31 12 3,4,5-trimethoxybenzoic acid 1 copy structure(N-termacetylated) 381.43 250.00 655.43 250.00 655.43 22 Gallic acid 4 copystructure 1008.98 17.61 17.45 2.86 2.84 17 Syringic acid 4 copystructure 1121.20 145.80 130.04 95.95 85.58 18 3,4-Dimethoxybenzoic acid4 copy structure 1057.20 111.30 105.28 90.21 85.33 214-ethoxycarbonyl-3,5-dimethoxybenzoic acid 4 copy structure 1121.19162.50 144.94 114.90 102.48 19 3,4,5-triethoxybenzoic acid 4 copystructure 1345.60 250.00 185.79 250.00 185.79 24 3,5-dihydroxybenzoicacid 4 copy structure 944.98 250.00 264.56 250.00 264.56 203,4,5-trimethoxybenzoic acid 4 copy structure 1177.30 250.00 212.35250.00 212.35 23 4-Hydroxybenzoic acid 4 copy structure 880.98 250.00283.77 250.00 283.77 30 Gallic acid Linear 3 copy structure 729.69 25.8935.48 8.36 11.45 26 3,4-Dimethoxybenzoic acid Linear 3 copy structure765.86 125.00 163.22 85.62 111.80 294-ethoxycarbonyl-3,5-dimethoxybenzoic acid Linear 3 copy structure813.85 111.50 137.00 93.43 114.80 27 3,4,5-triethoxybenzoic acid Linear3 copy structure 982.16 250.00 254.54 250.00 254.54 25 Syringic acidLinear 3 copy structure 813.86 250.00 307.18 250.00 307.18 283,4,5-trimethoxybenzoic acid Linear 3 copy structure 855.93 250.00292.08 250.00 292.08 31 4-Hydroxybenzoic acid Linear 3 copy structure633.69 250.00 394.51 250.00 394.51 32 3,5-dihydroxybenzoic acid Linear 3copy structure 681.69 250.00 366.74 250.00 366.74 374-ethoxycarbonyl-3,5-dimethoxybenzoic acid Linear 2 copystructure(N-term acetylated) 675.73 82.46 122.03 54.27 80.31 38 Gallicacid Linear 2 copy structure(N-term acetylated) 619.62 87.78 141.6754.27 87.59 33 Syringic acid Linear 2 copy structure(N-term acetylated)675.73 97.85 144.81 72.17 106.80 34 3,4-Dimethoxybenzoic acid Linear 2copy structure(N-term acetylated) 643.73 121.40 188.59 87.01 135.17 363,4,5-trimethoxybenzoic acid Linear 2 copy structure(N-term acetylated)703.78 250.00 355.22 125.00 177.61 39 4-Hydroxybenzoic acid Linear 2copy structure(N-term acetylated) 555.62 250.00 449.95 158.60 285.45 353,4,5-triethoxybenzoic acid Linear 2 copy structure(N-term acetylated)787.93 250.00 317.29 250.00 317.29 40 3,5-dihydroxybenzoic acid Linear 2copy structure(N-term acetylated) 587.62 250.00 425.45 250.00 425.45 46Gallic acid Linear 4 copy structure 1009.97 14.92 14.77 2.00 1.98 41Syringic acid Linear 4 copy structure 1122.19 89.77 80.00 83.26 74.19 423,4-Dimethoxybenzoic acid Linear 4 copy structure 1058.19 107.00 101.12113.80 107.54 45 4-ethoxycarbonyl-3,5-dimethoxybenzoic acid Linear 4copy structure 1122.18 154.20 137.41 136.30 121.46 433,4,5-triethoxybenzoic acid Linear 4 copy structure 1346.59 250.00185.65 250.00 185.65 48 3,5-dihydroxybenzoic acid Linear 4 copystructure 945.97 250.00 264.28 250.00 264.28 44 3,4,5-trimethoxybenzoicacid Linear 4 copy structure 1178.29 250.00 212.17 250.00 212.17 474-Hydroxybenzoic acid Linear 4 copy structure 881.97 250.00 283.46250.00 283.46 54 Gallic acid Linear 3 copy structure(N-term acetylated)899.90 6.85 7.61 3.00 3.33 50 3,4-Dimethoxybenzoic acid Linear 3 copystructure(N-term acetylated) 936.07 77.09 82.36 42.79 45.71 534-ethoxycarbonyl-3,5-dimethoxybenzoic acid Linear 3 copystructure(N-term acetylated) 1200.25 90.15 75.11 114.80 95.65 49Syringic acid Linear 3 copy structure(N-term acetylated) 984.07 118.00119.91 108.80 110.56 56 3,5-dihydroxybenzoic acid Linear 3 copystructure(N-term acetylated) 851.90 125.00 146.73 125.00 146.73 523,4,5-trimethoxybenzoic acid Linear 3 copy structure(N-term acetylated)1026.14 174.80 170.35 226.70 220.93 55 4-Hydroxybenzoic acid Linear 3copy structure(N-term acetylated) 803.90 250.00 310.98 250.00 310.98 513,4,5-triethoxybenzoic acid Linear 3 copy structure(N-term acetylated)1152.37 250.00 216.95 250.00 216.95IC 50 = concentration of compound at which inhibition of binding is 50%

A second batch of the MSI 27 library of compounds was synthesized andthe biotinylated UEA-1 binding inhibitory activity of compounds from thenew batch (called MSI 40) was compared with their chemically identicalcounterparts from MSI 27. As shown in Tables 7(a) and 7(b), chemicallyidentical compounds from both batches behaved in a similar manner. Inaddition, compounds from these libraries having gallic acid as theiractive binding moiety were compared with compounds where othercarboxylic acids were attached as side groups on the lysine linear orbranched scaffolds. As seen in Tables 7(a), (b) compounds having gallicacid as their side groups had low IC 50 values which suggested that theUEA-1 receptor binding inhibitory activity of these compounds wasrelated to the specific gallic acid structure rather than being ageneral feature of carboxylic acids.

From these data, it appears that having more than one polyhydroxy arylgroup contributes to good inhibition. Therefore, a range of suchcompounds was synthesized where up to eight—it could of course bemore—galloyl polyhydroxy side groups were attached to lysine and otherscaffolds, examples of which are shown below. The inhibitory activity ofthese compounds is shown in Table 8. TABLE 8 Structure Synthesis NumberIC 50 (ug/ml) IC 50 (uM) One copy N-Acetylated

MST 27 #14 1^(st) tier = 72 2^(nd) tier = 211 1^(st) tier = 54 2^(nd)tier = 160 Two copy branched

MSI 27 #6 MSI 40 #1  1^(st) tier = 11 2^(nd) tier = 8 1^(st) tier = 242^(nd) tier = 17 Two copy DAP

MSI 34 #1 MSI 39 #1  1^(st) tier = 1 2^(nd) tier = 0.8 1^(st) tier = 42^(nd) tier = 2 Two copy DAB

MSI 34 #2 MSI 39 #5  1^(st) tier = 3 2^(nd) tier = 2 1^(st) tier = 62^(nd) tier = 5 Two copy Orn

MSI 34 #3  1^(st) tier = 2 2^(nd) tier = 0.7 1^(st) tier = 4 2^(nd) tier= 2 Two copy Phe (p-NH2)

MSI 34 #4  1^(st) tier = 17 2^(nd) tier = 18 1^(st) tier = 35 2^(nd)tier = 37 Two copy N-Acetylated

MSI 27 #38 1^(st) tier = 54 2^(nd) tier = 48 1^(st) tier = 86 2^(nd)tier = 77 Three copy linear

MSI 27 #30 MSI 39 #9  1^(st) tier = 14 2^(nd) tier = 5 1^(st) tier = 192^(nd) tier = 7 Three copy linear DAP

MSI 34 #9  1^(st) tier = 0.6 2^(nd) tier = 0.5 1^(st) tier = 0.9 2^(nd)tier = 0.8 Three copy linear DAB

MSI 34 #10 1^(st) tier = 0.6 2^(nd) tier = 0.7 1^(st) tier = 0.9 2^(nd)tier = 1 Three copy linear Orn

MSI 34 #11 1^(st) tier = 1 2^(nd) tier = 0.8 1^(st) tier = 2 2^(nd) tier= 1 Three copy linear Phe (p-NH2)

MSI 34 #12 1^(st) tier = 6 2^(nd) tier = 4 1^(st) tier = 8 2^(nd) tier =5 Three copy N-Acetylated

MSI 27 #54 MSI 39 #10 1^(st) tier = 4 2^(nd) tier = 2 1^(st) tier = 52^(nd) tier = 3 Three copy N-Ac DAP

MSI 34 #13 1^(st) tier = 2 2^(nd) tier = 2 1^(st) tier = 3 2^(nd) tier =2 Three copy N-Ac DAB

MSI 34 #13 1^(st) tier = 2 2^(nd) tier = 1 1^(st) tier = 2 2^(nd) tier =1 Three copy N-Ac Orn

MSI 34 #15 1^(st) tier = 8 2^(nd) tier = 2 1^(st) tier = 9 2^(nd) tier =2 Three copy N-Ac Phe (p-NH2)

MSI 34 #16 1^(st) tier = 3 2^(nd) tier = 6 1^(st) tier = 3 2^(nd) tier =6 Four copy branched

MSI 27 #22 MSI 40 #8  1^(st) tier = 10 2^(nd) tier = 1 1^(st) tier = 102^(nd) tier = 1 Four copy DAP

MSI 34 #5  1^(st) tier = 0.5 2^(nd) tier = 1 1^(st) tier = 0.5 2^(nd)tier = 1 Two-by-Two DAP with linker

MSI 39 #4  1^(st) tier = 6 2^(nd) tier = 0.8 1^(st) tier = 5 2^(nd) tier= 0.7 Four copy DAB

MSI 34 #6  1^(st) tier = 0.6 2^(nd) tier = 1 1^(st) tier = 0.6 2^(nd)tier = 1 Four copy Orn

MSI 34 #7  1^(st) tier = 0.9 2^(nd) tier = 1 1^(st) tier = 0.9 2^(nd)tier = 1 Four copy Phe (p-NH2)

MSI 34 #8  1^(st) tier = 4 2^(nd) tier = 3 1^(st) tier = 4 2^(nd) tier =2 Four copy linear

MSI 27 #46 MSI 39 #11 1^(st) tier = 8 2^(nd) tier = 0.6 1^(st) tier = 82^(nd) tier = 0.6 Four copy linear DAP

MSI 34 #17 1^(st) tier = 1 2^(nd) tier = 0.7 1^(st) tier = 1 2^(nd) tier= 0.8 Four copy linear DAB

MSI 34 #18 1^(st) tier = 3 2^(nd) tier = 0.9 1^(st) tier = 3 2^(nd) tier= 1 Four copy linear Orn

MSI 34 #19 1^(st) tier = 11 2^(nd) tier = 1 1^(st) tier = 11 2^(nd) tier= 1 Four copy linear Phe (p-NH2)

MSI 34 #20 1^(st) tier = 2 2^(nd) tier = 6 1^(st) tier = 2 2^(nd) tier =5 Four copy N-Acetylated

MSI 30 #5 MSI 39 #12 1^(st) tier = 8 2^(nd) tier = 3 1^(st) tier = 72^(nd) tier = 2 Four copy N-Ac DAP

MSI 34 #21 1^(st) tier = 2 2^(nd) tier = 1 1^(st) tier = 2 2^(nd) tier =1 Four copy N-Ac DAB

MSI 34 #22 1^(st) tier = 3 2^(nd) tier = 1 1^(st) tier = 3 2^(nd) tier =1 Four copy N-Ac Orn

MSI 34 #23 1^(st) tier = 6 2^(nd) tier = 1 1^(st) tier = 5 2^(nd) tier =1 Four copy N-Ac Phe (p-Nh2)

MSI 34 #24 1^(st) tier = 3 2^(nd) tier = 9 1^(st) tier = 2 2^(nd) tier =7 Five copy linear

MSI 30 #5  1^(st) tier 75 2^(nd) tier = 12 1^(st) tier = 64 2^(nd) tier= 10 Five copy N-Acetylated

MSI 30 #8  1^(st) tier = 70 2^(nd) tier = 15 1^(st) tier = 48 2^(nd)tier = 10 Six copy linear

MSI 30 #10 1^(st) tier = 23 2^(nd) tier = 5 1^(st) tier = 14 2^(nd) tier= 3 Six copy N-Acetylated

MSI 30 #11 1^(st) tier = 13 2^(nd) tier = 2 1^(st) tier = 7 2^(nd) tier= 1 Seven copy linear

MSI 30 #13 1^(st) tier = 9 2^(nd) tier = 3 1^(st) tier = 5 2^(nd) tier =1 Seven copy N-Acetylated

MSI 30 #14 1^(st) tier = 17 2^(nd) tier = 9 1^(st) tier = 8 2^(nd) tier= 4 Eight copy linear

MSI 30 #16 1^(st) tier = 19 2^(nd) tier = 8 1^(st) tier = 9 2^(nd) tier= 4 Eight copy branched

MSI 30 #17 1^(st) tier = 5 2^(nd) tier = 5 1^(st) tier = 2 2^(nd) tier =2

Note:The Lysine scaffolds are synthesized from Fmoc-D-Lys(Boc)-OH whereasother scaffolds (DAP, DAB etc) are synthesized from the L derivatives.R = 3,4,5-trihydroxybenzoylPharmaceutical Formulations

As regards the kinds of pharmaceutical formulations to which theinvention relates, it will be understood from the above that they may ingeneral be any kind of formulation which is to be applied to the body'sepithelium, but more particularly will generally be enteric formulationsand most preferably oral formulations. Typically these consist of orcomprise solids, such as capsules, tablets, powders, emulsions, such asmicroemulsions and other types thereof and suspensions. Preferredembodiments include controlled release oral formulations, in which thepharmaceutically-active ingredient is encapsulated in a biodegradablepolymeric body or coating, e.g. by means of a solvent evaporationmethod. Coatings of this kind are known in the art, for example thepolylactide polymer coatings discussed in our WO-A-00/12124 andelsewhere. There is no particular limit on the type of biodegradablepolymer that may be used.

The encapsulated pharmaceutically-active material is preferably in theform of small particles, particularly microparticles or nanoparticles.For example, it may be a particulate formulation in which at least 50%of the particles are smaller than 5 μm, or more preferably in which asleast 50% of the particles are smaller than 600 nm. Microparticulate andnanoparticulate compositions of this type, comprising drug-activematerial encapsulated in biodegradable polymer, are known as such to theskilled person: see e.g. WO-A-00/12124 and WO-A-96/31202.

The present ligand compound may be bound to, coated onto or blended withthe drug formulation in any appropriate manner using physical andchemical techniques appropriate to the compounds concerned. Typicallythese consist of, but are not limited to, passive adsorption, directconjugation during one step synthesis (e.g. ligand-peptide drugsynthesis on standard columns or in solution), covalent coupling (e.g.amino group of ligand to carboxylate modified drug or delivery systemusing standard methodologies such as carbodiimide), andbiotin-streptavidin interaction (e.g. using biotinylated ligand andstreptavidin modified drug or delivery system).

As is also known practice, the particulate formulation may be given an“enteric coating” to protect it against gastric fluids so that theparticles can pass intact into the intestine.

Development of a Whole Cell Binding Assay for the Characterization ofBinding Affinities of the Lectin Mimetics:

A whole cell binding assay was developed for the characterization ofbinding affinities of various lectin mimetics of UEA-1. This assay wasdeveloped to allow structure activity analysis of these UEA-1 mimeticsin order to identify functional groups that enhance activity using wholecells in solution.

Methods:

Caco-2 cells were analysed by flow cytometry for binding of bothbiotinylated UEA-1 and a biotinylated lectin mimetic of UEA-1 using astreptavidin FITC probe. While clear binding (positives) of biotinylatedUEA-1 at concentrations as low as 1.0 μg/ml was seen, binding of thelectin mimetics of UEA-1 was negative even at concentrations of up to 65μM. In order to amplify the signal, a FITC-avidin D sandwich protocolwas used. In this method, after binding of the biotinylated compounds tothe cells, a series of FITC-avidin D/anti-avidin D/FITC-avidin Dstainings were performed, which resulted in a several fold increase influorescent signal.

Results:

The sandwich protocol described above increased the fluorescent signalas evidenced by the ability to measure a biotinylated UEA-1 sample of0.02 μg/ml. Previously, the lower limit had been 1.0 μg/ml with thestreptavidin-FITC probe. The nucleic acid stain 7-actinomycin D (7-AAD),a fluorescent dye for dead cells, was used to exclude these cells fromanalysis as they were known to bind non specifically to FITC-avidin D.Binding of biotinylated lectin mimetics of UEA-1 was demonstrated atboth 50 μm and 10 μm concentrations.

Approximately, 15% of the population of Caco-2 cells tested were FL1(FITC) positive, FL3(7-AAD)negative at both concentrations aftersubtraction of background fluorescence. The streptavidin FITC stain wasunable to detect these concentrations of the compound.

CONCLUSION

A whole cell binding assay has been developed for the characterizationof binding affinities of small molecule lectin mimetics of UEA-1. Thiswill allow structure activity analysis of these mimetics to identifythose functional groups that enhance activity using whole cells insolution. This assay will aid selection of small molecule mimics of UEA1for further studies in vivo and is an independent novel aspect herein.

Evaluation of the Ability of Gallic Acid UEA 1 Mimetic Compounds toMediate Delivery of a Model Particle System In Vivo:

The ability of compounds having gallic acid side chains to mimic UEA-1was assessed in vivo in mouse models. Here, binding and uptake by Mcells of particles coated with these compounds was assessed.

Methods:

Biotinylated ligand MSI 35-2 (4 copies of gallic acid; lysine scaffold)(see FIG. 5), a UEA-1 control (Vector Laboratories) and a biocytincontrol (Molecular Probes) were adsorbed on to fluorescent Estaporstreptavidin polystyrene particles (FITC label; 0.289 μm diameter) usingroutine methodologies at room temperature.

Mouse intestinal loops containing one or more Peyer's Patches wereinoculated with polystyrene particle suspensions (typically 500 μlcontaining 5.0×10¹⁰ particles per ml) and incubated for 30 min. ThePeyer's patches were excised, fixed in methanol and the M cells werecounter-stained with UEA1-rhodamine for subsequent analysis by confocalmicroscopy. Stained tissues were examined on a BioRad MRC 600 confocallaser scanning microscope equipped with an argon/krypton mixed gaslaser. The number of particles observed per total area was measured andreported quantitatively. For details of typical procedures and fullprotocols see: Foster, N., Clark, M. A., Jepson, M. A. & Hirst, B. H.Ulex europaeus 1 lectin targets microspheres to mouse Peyer's patchM-cells in vivo. Vaccine 1998:16(5); 536-541.

Results:

Scatter plots (log scale) for binding and uptake of MSI35-2, UEA1 andcontrol coated particles are illustrated as FIGS. 2-4.

-   FIG. 2 Surface binding and uptake of MSI35-2 gallic acid mimetic    coated particles.-   FIG. 3 Surface binding and uptake of UEA1 coated particles.-   FIG. 4 Surface binding & uptake of biocytin mimetic coated    particles.-   Each point on the scatter plot represents one image.

CONCLUSIONS

Fluorescent streptavidin polystyrene particles coated with thebiotinylated ligand MSI35-2 (4 copies of gallic acid; lysine scaffold)exhibited binding and uptake into M-cells in vivo comparable to orgreater than UEA-1 coated control particles in a mouse intestinal loopmodel. Binding and uptake into M-cells was significantly higher thanthat observed using biocytin coated control particles.

The potential for use of these lectin mimetics in oral targeted drugdelivery applications has been demonstrated using a model particulatesystem. The M-cell specific nature of the mimetic in the mouseintestinal loop model is of particular interest in the context ofvaccine delivery to antigen presenting cells.

Staining of Human Tissue Sections with UEA-1:

The application of UEA-1 as a diagnostic/prognostic indicator of diseasestates was verified in human tissues.

Methods:

I. Immunohistochemistry Procedure

Using UEA1 @ 0.25 μg/ml, goat anti-UEA1 @ 1:10,000 dilution and a VectorABC-AP Kit (AK5002) with a Vector Red substrate kit (SK-5100)microscopic analysis of human tissue sections revealed a fuchsia-coloredred deposit at the site of ligand binding. Negative controls, performedin the absence of the primary ligand prior to application of the goatanti-UEA1 antibody and detection staining, revealed negligiblebackground. Tissues stained with a positive control antibody (i.e. CD31;to ensure that the tissue antigens were preserved and accessible forimmunohistochemical analysis) confirmed tissue integrity. II. TissueSources and Diagnoses Tissue Diagnosis Sample ID Age/Sex Ileum Normal,with Peyer's 1 68 F patches Ileum Normal, with Peyer's 2 36 F patchesColon Normal 1 26 M Colon Normal 2 73 F Colon Carcinoma 1 Adult ColonCarcinoma 2 Adult Colon Crohn's disease 1 25 M Colon Crohn's disease 231 M Colon Ulcerative colitis 1 Adult Colon Ulcerative colitis 2 54 MResults:

-   FIGS. 6, 7 show images of the stained cell samples. In the normal    tissue, both the ileum and colon mucosal epithelia showed moderately    positive cytoplasmic labeling of the absorptive, goblet, M-cell, and    crypt enterocytes with UEA1. The staining reaction was slightly more    pronounced in the colon than in the ileum and appeared strongest    within cytoplasmic vacuoles, especially along the microvillus    luminal surface of the mucosal cells. The stained material was    secreted into the mucous that lines the intervillous and surface    mucosal cells. Cell membranes and/or glycocalyx were stained, while    the intercellular junctions were not demonstrated by the staining of    the ligand binding sites. Goblet cells in both samples showed    staining of fine dust-like to punctate cytoplasmic vesicles, and    almost all goblet cells had strong staining of globular    proteinaceous masses within the large cytoplasmic mucous vacuole.    The staining indicated that the ligand binds to a specific component    in the mucous or at the apical-mucous interface on the apical    surface and not mucous in general, because some of the goblet cells,    which contained mucous, did not stain. Several macrophages were    positive with what appeared to be phagocytized debris.

Throughout the stroma of the normal and diseased ileum and colon and thecolon carcinoma, there was a moderate to marked staining of all of theendothelial cells, and a minimal to moderate staining of the Schwanncells surrounding nerve axons. The Meissner's plexus and myentericplexus and their axons were consistently positive.

In the colon carcinoma samples the intensity of staining was similar tothat of normal colon. However, the pattern was somewhat different. Inthe colon carcinoma sections the foci of cytoplasmic staining tended tobe smaller and of more uniform size and cytoplasmic distribution. In thenormal tissue the reactivity was marked in the goblet cells, whereas inthe carcinoma tissue goblet cells were less common and did not stain asintensely. In the colon sections, the staining reactivity grade wassimilar to that of the normal samples, but it was noticeably higher thanthat in the adjacent normal colon tissue in the same sample as theevaluated neoplastic tissue.

The immunohistological evaluation of inflammatory bowel disease with theligand consisted of staining two cases each of Crohn's disease andulcerative colitis. The expression of UEA1 receptors appeared to besignificantly up-regulated in all of the mucosal enterocyte types inCrohn's disease, and appeared to be significantly down-regulated inulcerative colitis in all of the colonic mucosal enterocytes.

CONCLUSIONS

This difference in intensity of UEA-1 staining and/or patterning of thestaining as between normal and afflicted cells provides a basis for adiagnosis or prognosis in the present techniques.

Further Staining of Human Tissue Sections with UEA-1 and Gallic AcidUEA-1 Mimetic Compounds:

Staining of normal human tissues with UEA-1 and a gallic acid UEA-1mimetic was compared as an initial step to determine if the mimeticwould also be suitable for use as a diagnostic/prognostic indicator ofdisease states in human tissues.

Methods:

UEA1 and the biotinylated ligand MSI 35-2 (4 copies of gallic acid;lysine scaffold) (see FIG. 5) were used in staining protocols comparableto that described above. Negative controls, performed in the absence ofthe primary ligand, revealed negligible background.

Results:

Table 9 summarises the normal human tissue staining profiles for UEA-1and the UEA-1 mimetic. TABLE 9 UEA-1 UEA-1 mimetic Oesophagus +++ −Stomach +++ − Small intestine +++ ++ Large intestine +++ +++ Pancreas+++ − Muscle +/− − Brain ++ − Kidney +++ − Liver +++ − Spleen +++ −

CONCLUSIONS

The staining profile observed using the UEA-1 mimetic on large intestinesections was comparable to that obtained using UEA-1 (as describedabove). This correlated well with the selection procedure for the UEA-1mimetics, which was carried out using human Caco-2 cell membranefractions i.e. cells which exhibit features characteristic of colonicepithelia. The UEA-1 mimetic also exhibited staining of small intestinesections as would be expected. Interestingly, no gastrointestinal orother organ tissue was stained using the UEA-1 mimetic. This was inmarked contrast to UEA-1, which stained all of the tissue types, and maybe a favourable factor in selection of the mimetic over UEA-1 forclinical applications.

Evaluation of Binding and Uptake of the Gallic Acid UEA-1 Mimetic intoEndothelial Cells:

The gallic acid UEA-1 mimetic was compared to known cell permeablepeptides in an endothelial cell permeability assay to assess it'sapplication in a) diagnosis/prognosis of disease states (by monitoringvascularization) and b) in therapeutic delivery of pharmaceuticalformulations.

Methods:

-   1. ECV-304 cells were seeded on round coverslips at a concentration    of 2×10⁵ cells per coverslip, on 24 well plates, and allowed to grow    to confluency.-   2. The serum-containing medium was replaced by serum-free medium    (OptiMEM with penicillin streptomycin).-   3. Aqueous stock solutions of ligands were added directly to the    medium surrounding the cells to reach a final concentration of 10    μM, in 500 μl of medium.-   4. One plate of samples was incubated at 37° C. in 5% CO₂ enriched    air, and the other at 4° C. in air, both plates were incubated for 1    hr.-   5.Coverslips were then given 3×5 minute washes with PBS.-   6.Cells were fixed and permeabilised in 500 μl of methanol at    −20° C. for 10 minutes.-   7. A further 3×5 minute washes with PBS were given.-   8.Non-specific binding sites were blocked by incubating the cells    for 1 hr at room temperature in 500 μl of 5% milk solution in PBS    (Marvel).-   9.The peptides were stained with 500 μl of 15 nM Streptavidin-FITC    for 1 hr at room temperature.-   10. Cells were given final 3×5 minute washes with PBS.-   11. The coverslips were then removed from the wells and dipped    briefly in water, before being mounted on glass slides with    Vectashield mounting medium for fluorescence with Dapi.    Results:

See FIG. 8 for comparison of the UEA-1 mimetic to a known cell permeablepeptide and relevant controls. The UEA-1 mimetic exhibited strongstaining of the nucleus with diffuse staining throughout the cytoplasm.

CONCLUSIONS

Uptake of the UEA-1 mimetic by endothelial cells is suggestive ofsuitability of the mimetic for use as a marker of endothelia/bloodvessels, and hence, as a diagnostic or prognostic marker in diseasestates.

In addition the uptake profile correlated well with that of the SynB1peptide which is known to mediate delivery of therapeutic agents such asdoxorubicin into cells. Further applications of the UEA-1 mimetic indelivery of pharmaceutical formulations through mechanisms involvingdirect interaction with lipid membranes (as in the case of SynB1) willbe investigated.

1. A pharmaceutical formulation comprising a pharmaceutical agent and abioadhesive ligand, said bioadhesive ligand comprising a organocyclicmoiety, said organocyclic moiety a polyhydroxy- orpolyalkoxy-substituted moiety.
 2. The pharmaceutical formulation ofclaim 1 wherein the bioadhesive ligand is covalently or noncovalentlybound to a carrier entity comprising the pharmaceutical agent.
 3. Thepharmaceutical formulation of claim 2, wherein the carrier entity isselected from the group consisting of a nanoparticle, a microparticle,and a liposome.
 4. The pharmaceutical formulation of claim 1 wherein thebackbone of the organocyclic moiety comprises a backbone ring thatconsists of 5 to 7 atoms.
 5. The pharmaceutical formulation of claim 4wherein the ring backbone of 5 to 7 atoms is unsaturated.
 6. Thepharmaceutical formulation of claim 4 wherein all the atoms of the ringbackbone are carbon atoms.
 7. The pharmaceutical formulation of claim 4where the backbone of the organocyclic moiety is identical to that of aradical selected from the group consisting of phenyl, napthyl,cyclohexyl, benzyl, benzoyl, pyridine, and dihydrobenzopyran.
 8. Thepharmaceutical formulation of claim 4 wherein the organocyclic moiety isone in which two or more hydroxyl groups are substituted in a radicalselected from the group consisting of phenyl, napthyl, cyclohexyl,benzyl, benzoyl, pyridine, and dihydrobenzopyran.
 9. The pharmaceuticalformulation of claim 8 wherein the 2 to 4 hydroxyls are substituted ineach ring backbone of the selected radical.
 10. The pharmaceuticalformulation of claim 8 wherein the selected radical is a galloylradical.
 11. The pharmaceutical formulation of claim 1 wherein theorganocyclic moiety is covalently linked to a scaffold moiety.
 12. Thepharmaceutical formulation of claim 11 wherein the bioadhesive ligandcomprises two or more organocyclic moieties linked by a scaffold moiety.13. The pharmaceutical formulation of claim 12 wherein the shortestring-to-ring length along the scaffold and between the two organocyclicmoieties is from 1 to 20 atoms.
 14. The pharmaceutical formulation ofclaims 11 wherein the scaffold moiety comprises a moiety selected fromthe group consisting of amino acids, guanidines, hydantoins,thiohydantoins, thioureas, cathechins, acylamines, dicyclicamines,tricyclicamines, and saccharides.
 15. The pharmaceutical formulation ofclaim 13, wherein the scaffold moiety comprises an amino acid.
 16. Thepharmaceutical formulation of claim 15 wherein a trihydroxyphenyl ortrimethoxyphenyl moiety is linked to the amino acid.
 17. Thepharmaceutical formulation of claims 15 wherein the amino acid islysine.
 18. The pharmaceutical formulation of claim 15 wherein thescaffold moiety comprises a peptide comprising at least 2 amino acids.19. The pharmaceutical formulation of claim 15 wherein a galloyl moietyis linked to both of the at least 2 amino acids.
 20. The pharmaceuticalformulation of claim 18 wherein the at least two amino acids are bothlysines.
 21. The pharmaceutical formulation of claim 14 wherein thescaffold moiety selected is an acylamine.
 22. The pharmaceuticalformulation of claim 21 wherein the acylamine is of the structureX—NH—(C═O)—Y, where X comprises a linear backbone comprising at leasttwo atoms selected from the group, C and N, and wherein Y comprises anorganocyclic moiety.
 23. The pharmaceutical formulation of claim 20wherein the organocylic moiety is linked to the (C═O) group of theacylamine either directly or by a linker moiety backbone that does notexceed 10 atoms.
 24. The pharmaceutical formulation of claim 22 whereinthe R group of at least one amino acid is linked to the X moiety of theacylamine, said amino acid selected from the group consisting ofD-Norleucine, L-norleucine, D-tyrosine, L-tyrosine, D-cyclohexylalanine,D-cyclohexylalanine, D-arginine, and L-arginine.
 25. The pharmaceuticalformulation of claim 22 wherein an organocyclic moiety is linked to theX moiety of the acyl amine, said organocyclic moiety selected from thegroup consisting of D-napthylmethyl, L-napthylmethyl, andL-p-chloro-benzyl.
 26. The pharmaceutical formulation of claim 22wherein the Y moiety is selected from the group consisting of3,4,5-trihydroxyphenyl, 3,4,5-trimethoxyphenyl, 4-biphenylmethyl, and4-ethyl-4-biphenylmethyl.
 27. The pharmaceutical formulation of claim 22wherein —(C═O)—Y is a galloyl group.
 28. The pharmaceutical formulationof claim 22 wherein X comprises 2 to 10 organocylic moieties, eachlinked to the linear backbone either directly or by a linker moietybackbone that does not exceed 10 atoms.
 29. The pharmaceuticalformulation of claim 1 wherein the bioadhesive ligand comprises acompound selected from the group consisting of those compounds specifiedin Tables 1, 2, 3, 4, 5, 6, 7A, 7B, and
 8. 30. The compound selectedfrom the group consisting of those compounds listed in Tables 1, 2, 3,4, 5, 6, 7, 7A, 7B, and
 8. 31-38. (canceled)